Objective This study systematically investigated the alleviating effects and underlying physio-molecular mechanisms of the THFE (Trichoderma hamatum fungi extract) on tomato growth under 100 mM NaCl stress, aiming to provide a theoretical basis and practical foundation for the root application of microbial-derived biostimulants in agricultural production.

Methods A potted experiment was conducted using seedlings of the tomato (Solanum lycopersicum L.) cultivar 'Fenniao' as test material. Four treatments were established: CK (water control), CF (water + 0.05 mg/L THFE extract), NaCl (100 mmol/L NaCl solution), and NF (100 mmol/L NaCl + 0.05 mg/L THFE extract). Treatments commenced at the five-true-leaf stage of tomato seedlings. During the treatment period, CK and CF groups were irrigated with 100 mL water, while NaCl and NF groups received 100 mL of 100 mmol/L NaCl solution, after 48 h, CK and NaCl groups were supplemented with 100 mL water, whereas CF and NF groups were irrigated with 100 mL of 0.05 mg/L THFE extract. The process was considered as one treatment cycle, and a total of three treatment cycles were performed over eleven days. Then the measurements were taken immediately, including determination of tomato plant growth, photosynthetic parameters, and fruit yield; analysis of proline and catalase (CAT) content; observation of membrane permeability and cell death via DAB and Trypan Blue staining; analysis of expression changes in ABA signaling pathway and key proline biosynthesis genes; and characterization of THFE components using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS).

Results Compared with CK, NaCl treatment reduced tomato plant height, stem diameter, and biomass by more than 9.1%, 10.8%, and 30.5%, respectively, and decreased inflorescence number and flower number by 35.0% and 63.6%, respectively. Net photosynthetic rate and fruit yield were also reduced under salt stress. Salt stress induced reactive oxygen species burst, increasing malondialdehyde (MDA) content and relative electrolyte leakage by 155.1% and 185.5%, respectively, and significantly aggravating cell death. In contrast, the NF treatment restored vegetative growth indices, improved photosynthetic performance, and recovered fruit yield to near-control levels. Additionally, NF treatment activated osmoregulation and antioxidant systems, further enhancing proline content and catalase activity compared with NaCl treatment. At the molecular level, NF treatment down regulated the expression of ABA receptor gene SlPYR/PYL while greatly up regulated the proline biosynthesis genes SlP5CS1 and SlP5CS2. The THFE extract was characterized as a complex mixture comprising thousands of organic molecules, including amino acids and their derivatives, lipids, benzene derivatives, organic acids and their derivatives, among others.

Conclusion Salt stress significantly up-regulates the expression of SlPYR/PYL, SlP5CS1, and SlP5CS2 genes, triggering a reactive oxygen species (ROS) burst that leads to substantial H₂O₂ accumulation, exacerbated lipid peroxidation, and cell death. Compared to salt stress alone, the trichoderma hamatum extract (THFE) further enhances the expression of SlP5CS1 and SlP5CS2, strengthening proline biosynthesis and antioxidant enzyme activities in tomato. These effects synergistically maintain osmotic balance and redox homeostasis, mitigate membrane damage, and subsequently promote vegetative and reproductive development, ultimately improving the salt tolerance of tomato plants..